Search

The GGGGCC hexanucleotide expansion in C9orf72 (C9) is the most frequent known cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), yet a clear understanding of how C9 fits into the broader context of ALS/FTD pathology has remained lacking. The repetitive RNA derived from the C9 repeat is known to sequester hnRNPH, a splicing regulator, into insoluble aggregates, resulting in aberrant alternative splicing. Furthermore, hnRNPH insolubility and altered splicing of a robust set of targets have been observed to correlate in C9 and sporadic ALS/FTD patients alike, suggesting that changes along this axis are a core feature of disease pathogenesis. Here, we characterize previously uncategorized RNA splicing defects involving widespread intron retention affecting almost 2000 transcripts in C9ALS/FTD brains exhibiting a high amount of sequestered, insoluble hnRNPH. These intron retention events appear not to alter overall expression levels of the affected transcripts but rather the protein-coding regions. These retained introns affect transcripts in multiple cellular pathways predicted to be involved in C9 as well as sporadic ALS/FTD etiology, including the proteasomal and autophagy systems. The retained intron pre-mRNAs display a number of characteristics, including enrichment of hnRNPH-bound splicing enhancer motifs and a propensity for G-quadruplex (G-Q) formation, linking the defective splicing directly to high amounts of sequestered hnRNPH. Together, our results reveal previously undetected splicing defects in high insoluble hnRNPH-associated C9ALS brains, suggesting a feedback between effective RNA-binding protein dosage and protein quality control in C9, and perhaps all, ALS/FTD.

Peer reviewed: True, P elements were first discovered in the fruit fly Drosophila melanogaster as the causative agents of a syndrome of aberrant genetic traits called hybrid dysgenesis. This occurs when P element-carrying -males mate with females that lack P elements and results in progeny displaying sterility, mutations and chromosomal rearrangements. Since then numerous genetic, developmental, biochemical and structural studies have culminated in a deep understanding of P element transposition: from the cellular regulation and repression of transposition to the mechanistic details of the transposase nucleoprotein complex. Recent studies have revealed how piwi-interacting small RNA pathways can act to control splicing of the P element pre-mRNA to modulate transposase production in the germline. A recent cryo-electron microscopy structure of the P element transpososome reveals an unusual DNA architecture at the transposon termini and shows that the bound GTP cofactor functions to position the transposon ends within the transposase active site. Genome sequencing efforts have shown that there are P element transposase-homologous genes (called THAP9) in other animal genomes, including humans). This review highlights recent and previous studies, which together have led to new insights, and surveys our current understanding of the biology, biochemistry, mechanism and regulation of P element transposition.

Alternative premessenger RNA (pre-mRNA) splicing is a post-transcriptional mechanism for controlling gene expression. Splicing patterns are determined by both RNA-binding proteins and nuclear pre-mRNA structure. Here, we analyzed pre-mRNA splicing patterns, RNA-binding sites, and RNA structures near these binding sites coordinately controlled by two splicing factors: the heterogeneous nuclear ribonucleoprotein hnRNPA1 and the RNA helicase DDX5. We identified thousands of alternative pre-mRNA splicing events controlled by these factors by RNA sequencing (RNA-seq) following RNAi. Enhanced cross-linking and immunoprecipitation (eCLIP) on nuclear extracts was used to identify protein–RNA-binding sites for both proteins in the nuclear transcriptome. We found a significant overlap between hnRNPA1 and DDX5 splicing targets and that they share many closely linked binding sites as determined by eCLIP analysis. In vivo SHAPE (selective 2′-hydroxyl acylation analyzed by primer extension) chemical RNA structure probing data were used to model RNA structures near several exons controlled and bound by both proteins. Both sequence motifs and in vivo UV cross-linking sites for hnRNPA1 and DDX5 were used to map binding sites in their RNA targets, and often these sites flanked regions of higher chemical reactivity, suggesting an organized nature of nuclear pre-mRNPs. This work provides a first glimpse into the possible RNA structures surrounding pre-mRNA splicing factor-binding sites.

In Drosophila , P-element transposition causes mutagenesis and genome instability during hybrid dysgenesis. The P-element 31-bp terminal inverted repeats (TIRs) contain sequences essential for transposase cleavage and have been implicated in DNA repair via protein–DNA interactions with cellular proteins. The identity and function of these cellular proteins were unknown. Biochemical characterization of proteins that bind the TIRs identified a heterodimeric basic leucine zipper (bZIP) complex between an uncharacterized protein that we termed “Inverted Repeat Binding Protein (IRBP) 18” and its partner Xrp1. The reconstituted IRBP18/Xrp1 heterodimer binds sequence-specifically to its dsDNA-binding site within the P-element TIRs. Genetic analyses implicate both proteins as critical for repair of DNA breaks following transposase cleavage in vivo. These results identify a cellular protein complex that binds an active mobile element and plays a more general role in maintaining genome stability.

Alternative pre-mRNA splicing (AS) is a critical regulatory mechanism that operates extensively in the nervous system to produce diverse protein isoforms. Fruitless AS isoforms have been shown to influence male courtship behavior, but the underlying mechanisms are unknown. Using genome-wide approaches and quantitative behavioral assays, we show that the P-element somatic inhibitor (PSI) and its interaction with the U1 small nuclear ribonucleoprotein complex (snRNP) control male courtship behavior. PSI mutants lacking the U1 snRNP-interacting domain (PSIΔAB mutant) exhibit extended but futile mating attempts. The PSIΔAB mutant results in significant changes in the AS patterns of ∼1,200 genes in the Drosophila brain, many of which have been implicated in the regulation of male courtship behavior. PSI directly regulates the AS of at least one-third of these transcripts, suggesting that PSI-U1 snRNP interactions coordinate the behavioral network underlying courtship behavior. Importantly, one of these direct targets is fruitless, the master regulator of courtship. Thus, PSI imposes a specific mode of regulatory control within the neuronal circuit controlling courtship, even though it is broadly expressed in the fly nervous system. This study reinforces the importance of AS in the control of gene activity in neurons and integrated neuronal circuits, and provides a surprising link between a pleiotropic pre-mRNA splicing pathway and the precise control of successful male mating behavior.

Alternative pre-mRNA splicing (AS) greatly diversifies metazoan transcriptomes and proteomes and is crucial for gene regulation. Current computational analysis methods of AS from Illumina RNA-sequencing data rely on preannotated libraries of known spliced transcripts, which hinders AS analysis with poorly annotated genomes and can further mask unknown AS patterns. To address this critical bioinformatics problem, we developed a method called the junction usage model (JUM) that uses a bottom-up approach to identify, analyze, and quantitate global AS profiles without any prior transcriptome annotations. JUM accurately reports global AS changes in terms of the five conventional AS patterns and an additional “composite” category composed of inseparable combinations of conventional patterns. JUM stringently classifies the difficult and disease-relevant pattern of intron retention (IR), reducing the false positive rate of IR detection commonly seen in other annotation-based methods to near-negligible rates. When analyzing AS in RNA samples derived from Drosophila heads, human tumors, and human cell lines bearing cancer-associated splicing factor mutations, JUM consistently identified approximately twice the number of novel AS events missed by other methods. Computational simulations showed JUM exhibits a 1.2 to 4.8 times higher true positive rate at a fixed cutoff of 5% false discovery rate. In summary, JUM provides a framework and improved method that removes the necessity for transcriptome annotations and enables the detection, analysis, and quantification of AS patterns in complex metazoan transcriptomes with superior accuracy.

Although alternative pre-mRNA splicing (AS) significantly diversifies the neuronal proteome, the extent of AS is still unknown due in part to the large number of diverse cell types in the brain. To address this complexity issue, we used an annotation-free computational method to analyze and compare the AS profiles between small specific groups of Drosophila circadian neurons. The method, the Junction Usage Model (JUM), allows the comprehensive profiling of both known and novel AS events from specific RNA-seq libraries. The results show that many diverse and novel pre-mRNA isoforms are preferentially expressed in one class of clock neuron and also absent from the more standard Drosophila head RNA preparation. These AS events are enriched in potassium channels important for neuronal firing, and there are also cycling isoforms with no detectable underlying transcriptional oscillations. The results suggest massive AS regulation in the brain that is also likely important for circadian regulation., eLife digest The life of nearly all creatures on Earth follows the rhythm of day and night. For example, in fruit flies, darkness and light dictate when the insects feed, rest, move or mate. This is possible thanks to the circadian clock, an internal program which is synchronized with the environment to tell cells in the body when to perform certain roles. In fruit flies, the structure that keeps the body clock ticking is formed of about 150 ‘circadian neurons’, which are divided into several subgroups. In these cells, a complex genetic programis at work, with networks of genes being ‘switched on’ in a cyclical way. To understand how this program works, scientists need to know which genes are turned on and when, as well as which proteins are created based on the information contained in these genes. This can be difficult because one gene does not necessarily code for only one protein. Indeed, when a gene is turned on, it gets copied into a pre-messenger RNA (pre-mRNA), which is formed of several modules. The pre-mRNA can then go through a process called alternative splicing that shuffles or removes the different modules. This means that one gene can give rise to different pre-mRNA molecules that will each serve as a template to build a distinct protein. Until now, there have been few studies that examine the different types of pre-mRNAs found in circadian neurons, and how these change with the time of day. Here, Wang, Abruzzi et al. extract three subgroups of circadian neurons, and one subgroup of non-circadian neurons, from the brain of fruit flies. The pre-mRNAs are isolated, and then a new computational method, called JUM, identifies, counts and categorizes the pre-mRNA molecules in the different groups of neurons. This analysis reveals hundreds of previously unknown pre-mRNA molecules, many of which differed extensively between the types of brain cells. When comparing circadian and non-circadian neurons, Wang, Abruzzi et al. show that the circadian cells had more pre-mRNAs that code for proteins that help the cell communicate with other neurons. Finally, many genes in the circadian neurons use alternative splicing to turn on different types of pre-mRNA molecules at different times of the day in a cyclical way; this suggests that these pre-mRNAs might be participating in the genetic circadian program. Many human disorders, such as certain forms of insomnia, emerge when the circadian clock is thrown off balance. The results reported by Wang, Abruzzi et al. show that alternative splicing may be an overlooked mechanism that shapes this complex program.

Alternative pre-mRNA splicing (AS) greatly diversifies metazoan transcriptomes and proteomes and is crucial for gene regulation. Current computational analysis methods of AS from Illumina RNA-seq data rely on pre-annotated libraries of known spliced transcripts, which hinders AS analysis with poorly annotated genomes and can further mask unknown AS patterns. To address this critical bioinformatics problem, we developed a method called the Junction Usage Model (JUM) that uses a bottom-up approach to identify, analyze and quantitate global AS profiles without any prior transcriptome annotations. JUM accurately reports global AS changes in terms of the five conventional AS patterns and an additional “Composite” category composed of inseparable combinations of conventional patterns. JUM stringently classifies the difficult and disease-relevant pattern of intron retention, reducing the false positive rate of IR detection commonly seen in other annotation-based methods to near negligible rates. When analyzing AS in RNA-samples derived from Drosophila heads, human tumors and human cell lines bearing cancer-associated splicing factor mutations, JUM consistently identified ~ twice the number of novel AS events missed by other methods. Computational simulations showed JUM exhibits a 1.2-4.8 times higher true positive rate at a fixed cut-off of 5% false discovery rate. In summary, JUM provides a new framework and improved method that removes the necessity for transcriptome annotations and enables the detection, analysis and quantification of AS patterns in complex metazoan transcriptomes with superior accuracy.

In the 20 years since the RNA journal was started there have been many exciting and unexpected findings made in the broad field of RNA biology, biochemistry, genetics and structural biology. Below, I will briefly describe a few of the many seminal discoveries.

RNA sequence elements involved in the regulation of pre-mRNA splicing have previously been identified in vertebrate genomes by computational methods. Here, we apply such approaches to predict splicing regulatory elements in Drosophila melanogaster and compare them with elements previously found in the human, mouse, and pufferfish genomes. We identified 99 putative exonic splicing enhancers (ESEs) and 231 putative intronic splicing enhancers (ISEs) enriched near weak 5′ and 3′ splice sites of constitutively spliced introns, distinguishing between those found near short and long introns. We found that a significant proportion (58%) of fly enhancer sequences were previously reported in at least one of the vertebrates. Furthermore, 20% of putative fly ESEs were previously identified as ESEs in human, mouse, and pufferfish; while only two fly ISEs, CTCTCT and TTATAA, were identified as ISEs in all three vertebrate species. Several putative enhancer sequences are similar to characterized binding-site motifs for Drosophila and mammalian splicing regulators. To provide additional evidence for the function of putative ISEs, we separately identified 298 intronic hexamers significantly enriched within sequences phylogenetically conserved among 15 insect species. We found that 73 putative ISEs were among those enriched in conserved regions of the D. melanogaster genome. The functions of nine enhancer sequences were verified in a heterologous splicing reporter, demonstrating that these sequences are sufficient to enhance splicing in vivo. Taken together, these data identify a set of predicted positive-acting splicing regulatory motifs in the Drosophila genome and reveal regulatory sequences that are present in distant metazoan genomes.

Alternative splicing of precursor mRNA (pre-mRNA) is a strategy employed by most eukaryotes to increase transcript and proteomic diversity. Many metazoan splicing factors are members of multigene families, with each member having different functions. How these highly related proteins evolve unique properties has been unclear. Here we characterize the evolution and function of a new Drosophila splicing factor, termed LS2 (Large Subunit 2), that arose from a gene duplication event of dU2AF50, the large subunit of the highly conserved heterodimeric general splicing factor U2AF (U2-associated factor). The quickly evolving LS2 gene has diverged from the splicing-promoting, ubiquitously expressed dU2AF50 such that it binds a markedly different RNA sequence, acts as a splicing repressor, and is preferentially expressed in testes. Target transcripts of LS2 are also enriched for performing testes-related functions. We therefore propose a path for the evolution of a new splicing factor in Drosophila that regulates specific pre-mRNAs and contributes to transcript diversity in a tissue-specific manner.

In Drosophila melanogaster, female-specific expression of Sex-lethal (SXL) and Transformer (TRA) proteins controls sex-specific alternative splicing and/or translation of a handful of regulatory genes responsible for sexual differentiation and behavior. Recent findings in 2009 by Telonis-Scott et al. document widespread sex-biased alternative splicing in fruitflies, including instances of tissue-restricted sex-specific splicing. Here we report results arguing that some of these novel sex-specific splicing events are regulated by mechanisms distinct from those established by female-specific expression of SXL and TRA. Bioinformatic analysis of SXL/TRA binding sites, experimental analysis of sex-specific splicing in S2 and Kc cells lines and of the effects of SXL knockdown in Kc cells indicate that SXL-dependent and SXL-independent regulatory mechanisms coexist within the same cell. Additional determinants of sex-specific splicing can be provided by sex-specific differences in the expression of RNA binding proteins, including Hrp40/Squid. We report that sex-specific alternative splicing of the gene hrp40/squid leads to sex-specific differences in the levels of this hnRNP protein. The significant overlap between sex-regulated alternative splicing changes and those induced by knockdown of hrp40/squid and the presence of related sequence motifs enriched near subsets of Hrp40/Squid-regulated and sex-regulated splice sites indicate that this protein contributes to sex-specific splicing regulation. A significant fraction of sex-specific splicing differences are absent in germline-less tudor mutant flies. Intriguingly, these include alternative splicing events that are differentially spliced in tissues distant from the germline. Collectively, our results reveal that distinct genetic programs control widespread sex-specific splicing in Drosophila melanogaster.

Guanosine triphosphate (GTP) binding and hydrolysis events often act as molecular switches in proteins, modulating conformational changes between active and inactive states in many signaling molecules and transport systems. The P element transposase of Drosophila melanogaster requires GTP binding to proceed along its reaction pathway, following initial site-specific DNA binding. GTP binding is unique to P elements and may represent a novel form of transpositional regulation, allowing the bound transposase to find a second site, looping the transposon DNA for strand cleavage and excision. The GTP-binding activity has been previously mapped to the central portion of the transposase protein; however, the P element transposase contains little sequence identity with known GTP-binding folds. To identify soluble, active transposase domains, a GFP solubility screen was used testing the solubility of random P element gene fragments in E. coli. The screen produced a single clone spanning known GTP-binding residues in the central portion of the transposase coding region. This clone, amino acids 275–409 in the P element transposase, was soluble, highly expressed in E.coli and active for GTP-binding activity, therefore is a candidate for future biochemical and structural studies. In addition, the chimeric screen revealed a minimal N-terminal THAP DNA-binding domain attached to an extended leucine zipper coiled-coil dimerization domain in the P element transposase, precisely delineating the DNA-binding and dimerization activities on the primary sequence. This study highlights the use of a GFP-based solubility screen on a large multidomain protein to identify highly expressed, soluble truncated domain subregions.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) have been traditionally seen as proteins packaging RNA nonspecifically into ribonucleoprotein particles (RNPs), but evidence suggests specific cellular functions on discrete target pre-mRNAs. Here we report genome-wide analysis of alternative splicing patterns regulated by four Drosophila homologues of the mammalian hnRNP A/B family (hrp36, hrp38, hrp40 and hrp48). Analysis of the global RNA binding distributions of each protein revealed both small and also extensively bound regions on target transcripts. A significant subset of RNAs were bound and regulated by more than one hnRNP protein, revealing a combinatorial network of interactions. In vitro RNA binding site selection experiments (SELEX) identified distinct binding motif specificities for each protein that were over-represented in their respective regulated and bound transcripts. These results indicate that individual heterogeneous ribonucleoproteins have specific affinities for overlapping, but distinct, populations of target pre-mRNAs controlling their patterns of RNA processing.

The polypyrimidine tract binding protein (PTB) binds pre-mRNAs to alter splice-site choice. We characterized a series of spliceosomal complexes that assemble on a pre-mRNA under conditions of either PTB-mediated splicing repression or its absence. In the absence of repression, exon definition complexes that were assembled downstream of the regulated exon could progress to pre-spliceosomal A complexes and functional spliceosomes. Under PTB-mediated repression, assembly was arrested at an A-like complex that was unable to transition to spliceosomal complexes. Trans-splicing experiments indicated that, even when the U1 and U2 small nuclear ribonucleoprotein particles (snRNPs) are properly bound to the upstream and downstream exons, the presence of PTB prevents the interaction of the two exon complexes. Proteomic analyses of these complexes provide a new description of exon definition complexes, and indicate that splicing regulators can act on the transition between the exon definition complex and an intron-defined spliceosome.

The Down syndrome cell adhesion molecule (Dscam) gene has essential roles in neural wiring and pathogen recognition in Drosophila melanogaster. Dscam encodes 38,016 distinct isoforms via extensive alternative splicing. The 95 alternative exons in Dscam are organized into clusters that are spliced in a mutually exclusive manner. The exon 6 cluster contains 48 variable exons and uses a complex system of competing RNA structures to ensure that only one variable exon is included. Here we show that the heterogeneous nuclear ribonucleoprotein hrp36 acts specifically within, and throughout, the exon 6 cluster to prevent the inclusion of multiple exons. Moreover, hrp36 prevents serine/arginine-rich proteins from promoting the ectopic inclusion of multiple exon 6 variants. Thus, the fidelity of mutually exclusive splicing in the exon 6 cluster is governed by an intricate combination of alternative RNA structures and a globally acting splicing repressor.

P transposable elements are one of the best studied eukaryotic mobile DNA elements in metazoans. This chapter focuses on the more recent developments in understanding the mechanism and specificity of P element transposition through detailed biochemical and genetic experiments. With the use of P element transformation to introduce the yeast site-specific Flip recombinase (FLP) and the Flip recombinase target (FRT) recombination sites into the Drosophila genome, it was shown that the FLP recombinase could catalyze DNA strand exchange between FRT sites. Subsequently, it was shown that strand exchange could occur between FRT sites on different chromosomes or sister chromatids. With the development of efficient single P element mutagenesis strategies, many laboratories have undertaken large-scale P element insertional mutation screens. The ability to stimulate recombination at P element sites has had several useful applications in Drosophila genetics. A system for purifying the P element transposase protein from Drosophila tissue culture cell nuclear extracts was developed by using a combination of conventional and affinity chromotography methods. It will be extremely important and interesting for the future to understand how P element mobility might be linked to the cell cycle and DNA repair checkpoints and how P element transposition might be influenced by extracellular stimuli.

Genetic analysis of the Drosophila Bloom's syndrome helicase homolog (mus309/DmBLM) indicates that DmBLM is required for the synthesis-dependent strand annealing (SDSA) pathway of homologous recombination. Here we report the first biochemical study of DmBLM. Recombinant, epitope-tagged DmBLM was expressed in Drosophila cell culture and highly purified protein was prepared from nuclear extracts. Purified DmBLM exists exclusively as a high molecular weight ( approximately 1.17 MDa) species, is a DNA-dependent ATPase, has 3'-->5' DNA helicase activity, prefers forked substrate DNAs and anneals complementary DNAs. High-affinity DNA binding is ATP-dependent and low-affinity ATP-independent interactions contribute to forked substrate DNA binding and drive strand annealing. DmBLM combines DNA strand displacement with DNA strand annealing to catalyze the displacement of one DNA strand while annealing a second complementary DNA strand.

© 2015 ABRF. The Encyclopedia of DNA Elements (ENCODE) Project aims to identify all functional sequence elements in the human genome sequence by use of high-throughput DNA/cDNA sequencing approaches. To aid the standardization, comparison, and integration of data sets produced from different technologies and platforms, the ENCODE Consortium selected several standard human cell lines to be used by the ENCODE Projects. The Tier 1 ENCODE cell lines include GM12878, K562, and H1 human embryonic stem cell lines. GM12878 is a lymphoblastoid cell line, transformed with the Epstein-Barr virus, that was selected by the International HapMap Project for whole genome and transcriptome sequencing by use of the Illumina platform. K562 is an immortalized myelogenous leukemia cell line. The GM12878 cell line is attractive for the ENCODE Projects, as it offers potential synergy with the International HapMap Project. Despite the vast amount of sequencing data available on the GM12878 cell line through the ENCODE Project, including transcriptome, chromatin immunoprecipitationsequencing for histone marks, and transcription factors, no small interfering siRNA-mediated knockdown studies have been performed in the GM12878 cell line, as cationic lipid-mediated transfection methods are inefficient for lymphoid cell lines. Here, we present an efficient and reproducible method for transfection of a variety of siRNAs into the GM12878 and K562 cell lines, which subsequently results in targeted protein depletion.

Splicing of the Drosophila P-element third intron (IVS3) is repressed in somatic tissues due to the function of an exonic splicing silencer (ESS) complex present on the 5′ exon RNA. To comprehensively characterize the mechanisms of this alternative splicing regulation, we used biochemical fractionation and affinity purification to isolate the silencer complex assembled in vitro and identify the constituent proteins by mass spectrometry. Functional assays using splicing reporter minigenes identified the proteins hrp36 and hrp38 and the cytoplasmic poly(A)-binding protein PABPC1 as novel functional components of the splicing silencer. hrp48, PSI, and PABPC1 have high-affinity RNA-binding sites on the P-element IVS3 5′ exon, whereas hrp36 and hrp38 proteins bind with low affinity to the P-element silencer RNA. RNA pull-down and immobilized protein assays showed that hrp48 protein binding to the silencer RNA can recruit hrp36 and hrp38. These studies identified additional components that function at the P-element ESS and indicated that proteins with low-affinity RNA-binding sites can be recruited in a functional manner through interactions with a protein bound to RNA at a high-affinity binding site. These studies have implications for the role of heterogeneous nuclear ribonucleoproteins (hnRNPs) in the control of alternative splicing at cis-acting regulatory sites.

In this protocol, an RNA sample, fractionated by gel electrophoresis, is transferred from the gel onto a membrane by capillary transfer. Short-wave UV light is used to fix the transferred RNA to the membrane. The membrane is then pretreated to block nonspecific probe-binding sites, and hybridization of the immobilized RNA to a 32P-labeled DNA or RNA probe specific for the mRNA of interest is performed. Finally, the membrane is washed and subjected to autoradiography or phosphorimaging. Because exposure to UV cross-links the RNA to the membrane, the membrane can be stripped and hybridized with other probes. The procedure is suitable for detecting poly(A)+-selected mRNA or mRNA in total cellular RNA if the target transcript is relatively abundant. Using DNA or RNA probes labeled to 1 × 108–10 × 108 cpm/µg, it should be possible to detect ∼5 pg of a specific RNA.

Faithful propagation of eukaryotic chromosomes usually requires that no DNA segment be replicated more than once during one cell cycle. Cyclin-dependent kinases (Cdks) are critical for the re-replication controls that inhibit the activities of components of the pre-replication complexes (pre-RCs) following origin activation. The origin recognition complex (ORC) initiates the assembly of pre-RCs at origins of replication and Cdk phosphorylation of ORC is important for the prevention of re-initiation. Here we show that Drosophila melanogaster ORC (DmORC) is phosphorylated in vivo and is a substrate for Cdks in vitro. Cdk phosphorylation of DmORC subunits DmOrc1p and DmOrc2p inhibits the intrinsic ATPase activity of DmORC without affecting ATP binding to DmOrc1p. Moreover, Cdk phosphorylation inhibits the ATP-dependent DNA-binding activity of DmORC in vitro, thus identifying a novel determinant for DmORC-DNA interaction. DmORC is a substrate for both Cdk2·cyclin E and Cdk1·cyclin B in vitro. Such phosphorylation of DmORC by Cdk2·cyclin E, but not by Cdk1·cyclin B, requires an “RXL” motif in DmOrc1p. We also identify casein kinase 2 (CK2) as a kinase activity in embryonic extracts targeting DmORC for modification. CK2 phosphorylation does not affect ATP hydrolysis by DmORC but modulates the ATP-dependent DNA-binding activity of DmORC. These results suggest molecular mechanisms by which Cdks may inhibit ORC function as part of re-replication control and show that DmORC activity may be modulated in response to phosphorylation by multiple kinases.

Distinct isoforms of spliceosomal RNAs may be involved in regulating pre-messenger RNA splicing in eukaryotic cells. During a large-scale effort to identify small noncoding RNAs in Drosophila, we isolated a U5 snRNA-like molecule containing a 5′ segment identical to that of the canonical (major) U5 snRNA but with a variant Sm binding site and a distinct 3′ hairpin sequence. Based on this finding, another six similar U5 snRNA-like sequences were identified within the Drosophila genome by sequence similarity to the invariant loop in the 5′ half of U5. Interestingly, although all of these variants are expressed in vivo, each shows a distinct temporal expression profile during Drosophila development, and one is expressed primarily in fly heads. The presence of these U5 snRNA variants within RNP particles suggests their role in splicing and implies a possible connection to regulation of developmental and tissue-specific gene expression.

The protein factor U2AF is an essential component required for pre-mRNA splicing. Mutations identified in the S. pombe large U2AF subunit were used to engineer transgenic Drosophila carrying temperature-sensitive U2AF large subunit alleles. Mutant recombinant U2AF heterodimers showed reduced polypyrimidine tract RNA binding at elevated temperatures. Genome-wide RNA profiling comparing wild-type and mutant strains identified more than 400 genes differentially expressed in the dU2AF50 mutant flies grown at the restrictive temperature. Surprisingly, almost 40% of the downregulated genes lack introns. Microarray analyses revealed that nuclear export of a large number of intronless mRNAs is impaired in Drosophila-cultured cells RNAi knocked down for dU2AF50. Immunopurification of nuclear RNP complexes showed that dU2AF50 associates with intronless mRNAs. These results reveal an unexpected role for the splicing factor dU2AF50 in the nuclear export of intronless mRNAs.

We have used genetic and microarray analysis to determine how ionizing radiation (IR) induces p53-dependent transcription and apoptosis in Drosophila melanogaster. IR induces MNK/Chk2-dependent phosphorylation of p53 without changing p53 protein levels, indicating that p53 activity can be regulated without an Mdm2-like activity. In a genome-wide analysis of IR-induced transcription in wild-type and mutant embryos, all IR-induced increases in transcript levels required both p53 and the Drosophila Chk2 homolog MNK. Proapoptotic targets of p53 include hid, reaper, sickle, and the tumor necrosis factor family member EIGER: Overexpression of Eiger is sufficient to induce apoptosis, but mutations in Eiger do not block IR-induced apoptosis. Animals heterozygous for deletions that span the reaper, sickle, and hid genes exhibited reduced IR-dependent apoptosis, indicating that this gene complex is haploinsufficient for induction of apoptosis. Among the genes in this region, hid plays a central, dosage-sensitive role in IR-induced apoptosis. p53 and MNK/Chk2 also regulate DNA repair genes, including two components of the nonhomologous end-joining repair pathway, Ku70 and Ku80. Our results indicate that MNK/Chk2-dependent modification of Drosophila p53 activates a global transcriptional response to DNA damage that induces error-prone DNA repair as well as intrinsic and extrinsic apoptosis pathways.

We have recently shown that a third of reliably-inferred alternative mRNA isoforms are candidates for nonsense-mediated mRNA decay (NMD), an mRNA surveillance system (Lewis et al., 2003; Proc. Natl Acad. Sci. USA, 100, 189–192). Rather than being translated to yield protein, these transcripts are expected to be degraded and may be subject to regulated unproductive splicing and translation (RUST). Our initial experimental studies are consistent with these predictions and suggest an unappreciated role for NMD in several human diseases. Contact: brenner@compbio.berkeley.edu *To whom correspondence should be addressed.

Precursor messenger RNA (pre-mRNA) splicing is a critical step in the posttranscriptional regulation of gene expression, providing significant expansion of the functional proteome of eukaryotic organisms with limited gene numbers. Split eukaryotic genes contain intervening sequences or introns disrupting protein-coding exons, and intron removal occurs by repeated assembly of a large and highly dynamic ribonucleoprotein complex termed the spliceosome, which is composed of five small nuclear ribonucleoprotein particles, U1, U2, U4/U6, and U5. Biochemical studies over the past 10 years have allowed the isolation as well as compositional, functional, and structural analysis of splicing complexes at distinct stages along the spliceosome cycle. The average human gene contains eight exons and seven introns, producing an average of three or more alternatively spliced mRNA isoforms. Recent high-throughput sequencing studies indicate that 100% of human genes produce at least two alternative mRNA isoforms. Mechanisms of alternative splicing include RNA–protein interactions of splicing factors with regulatory sites termed silencers or enhancers, RNA–RNA base-pairing interactions, or chromatin-based effects that can change or determine splicing patterns. Disease-causing mutations can often occur in splice sites near intron borders or in exonic or intronic RNA regulatory silencer or enhancer elements, as well as in genes that encode splicing factors. Together, these studies provide mechanistic insights into how spliceosome assembly, dynamics, and catalysis occur; how alternative splicing is regulated and evolves; and how splicing can be disrupted by cis- and trans-acting mutations leading to disease states. These findings make the spliceosome an attractive new target for small-molecule, antisense, and genome-editing therapeutic interventions.

This protocol describes the detection of small RNAs (∼10–200 nucleotides) by blot hybridization. The RNA samples, denatured in formamide, are separated by denaturing polyacrylamide gel electrophoresis. Because high-percentage polyacrylamide gels are required to separate RNAs in this size range, it is necessary to perform electrophoretic transfer to positively charged nylon membranes. After transfer, the immobilized RNAs are subjected to hybridization with a 32P-radiolabeled DNA or RNA probe and detected by phosphorimaging or autoradiography. This procedure is commonly used to detect small, U-rich spliceosomal small nuclear RNAs (snRNAs) and miRNAs. It should be possible also to detect most miRNAs using high-percentage (e.g., 15%) urea–polyacrylamide gel electrophoresis.

This protocol is used to label RNA molecules (in vitro–synthesized or in vivo–purified RNA molecules) that have free 3′-hydroxyl termini. The reaction is performed in 10 min using yeast poly(A) polymerase and 3′-deoxyadenosine 5′-[α-32P]triphosphate (cordycepin 5′-[α-32P]triphosphate), a chain-terminating nucleotide. At the end of the procedure, the reaction is desalted by gel filtration to remove any unincorporated nucleotides.

P element somatic inhibitor (PSI) is a 97-kDa RNA-binding protein with four KH motifs that is involved in the inhibition of splicing of the Drosophila P element third intron (IVS3) in somatic cells. PSI interacts with a negative regulatory element in the IVS3 5' exon. This element contains two pseudo-5' splice sites, termed F1 and F2. To identify high affinity binding sites for the PSI protein, in vitro selection (SELEX) was performed using a random RNA oligonucleotide pool. Alignment of high affinity PSI-binding RNAs revealed a degenerate consensus sequence consisting of a short core motif of CUU flanked by alternative purines and pyrimidines. Interestingly, this sequence resembles the F2 pseudo-5' splice site in the P element negative regulatory element. Additionally, a negative in vitro selection of PCR-mutagenized P element 5' exon regulatory element RNAs identified two U residues in the F1 and F2 pseudo-5' splice sites as important nucleotides for PSI binding and the U residue in the F2 region is a nearly invariant nucleotide in the consensus SELEX motif. The high affinity PSI SELEX sequence acted as a splicing inhibitor when placed in the context of a P element splicing pre-mRNA in vitro. Data from in vitro splicing assays, UV crosslinking and RNA-binding competition experiments indicates a strong correlation between the binding affinities of PSI for the SELEX sequences and their ability to modulate splicing of P element IVS3 in vitro.

By binding specific RNA transcripts, the Sex-lethal protein (SXL) governs sexual differentiation and dosage compensation in Drosophila melanogaster . To investigate the basis for RNA binding specificity, we determined the crystal structure of the tandem RNA recognition motifs (RRMs) of SXL. Both RRMs adopt the canonical RRM fold, and the 10-residue, interdomain linker shows significant disorder. In contrast to the previously determined structure of the two-RRM fragment of heterogeneous nuclear ribonucleoprotein Al, SXL displays no interdomain contacts between RRMs. These results suggest that the SXL RRMs are flexibly tethered in solution, and RNA binding restricts the orientation of RRMs. Therefore, the observed specificity for single-stranded, U-rich sequences does not arise from a predefined, rigid architecture of the isolated SXL RRMs.

The generation of functional mRNA in eukaryotes requires the accurate removal of noncoding regions (introns) from pre-mRNA by a process termed pre-mRNA splicing (14, 25). Splicing takes place in the spliceosome, a dynamic RNA-protein complex that assembles in a stepwise manner on the pre-mRNA (10, 14). The spliceosome is composed of small nuclear ribonucleoprotein particles (snRNPs) and extrinsic (non-snRNP) factors. The recognition of exon/intron boundaries, the splice sites, by the splicing apparatus is a critical step in processing of both constitutively and alternatively spliced pre-mRNAs. U1 snRNP defines the 5′ splice site, and U2 snRNP defines the branchpoint sequence (3, 10, 14, 17). Since in most cases the first AG dinucleotide downstream of the branchpoint is used as the 3′ splice site, by defining the branchpoint, U2 snRNP defines the 3′ splice site (18, 27). Targeting of U2 snRNP to the branch site requires the extrinsic splicing factor U2 snRNP auxiliary factor (U2AF). U2AF binds site specifically to the intron pyrimidine tract between the branchpoint sequence and 3′ splice site at an early step in spliceosome assembly and recruits U2 snRNP to the branch site (22, 29, 42). Regulation of 3′ splice site choice, both positive and negative, can be realized by influencing the pyrimidine tract binding of U2AF (33, 35, 45). Because U2AF is a major determinant in 3′ splice site selection, it has been the subject of extensive biochemical and genetic investigation. Human U2AF is a heterodimer composed of a 65-kDa large subunit (hU2AF65) and a 35-kDa small subunit (hU2AF35) (41). Both subunits are conserved in other organisms (40), and U2AF homologs have been identified in Drosophila melanogaster (9, 21), Schizosaccharomyces pombe (16, 36), and Caenorhabditis elegans (3a, 39). The Drosophila U2AF large (dU2AF50)- and small (dU2AF38)-subunit homologs are 50 and 38 kDa, respectively (9, 21). The U2AF large subunit contains three RNA recognition motifs (RRMs) and an amino-terminal arginine-serine-rich (RS) domain (42). The small subunit contains a highly degenerate RRM (pseudo-RRM) (2), two Zn2+ binding motifs (37), and a carboxyl-terminal RS domain and glycine-rich region (43). Both U2AF subunits are involved in recognition of the intron pyrimidine tract. The large subunit (hU2AF65 and dU2AF50) is required for site-specific pyrimidine tract binding (9, 42). The small subunit acts as a cofactor to stabilize the large subunit on the pyrimidine tract, apparently through protein-protein interactions with constitutive and alternative splicing factors (38, 45). While it has been firmly established that all three RRMs on hU2AF65 are necessary for high-affinity RNA binding, a role for the large-subunit RS domain in RNA binding remains unresolved (11, 42). In one study removal of the hU2AF65 RS domain had a modest effect on RNA binding (42). In a second study, the RS domain was found to be absolutely required for RNA binding (11). In vitro splicing assays using U2AF-depleted extracts prepared by two independent methods have identified independent and essential roles for the two U2AF RS domains: the large-subunit RS domain is required to target U2 snRNP to the branch site (34, 42), and in the immunodepleted extracts under certain conditions, the small-subunit RS domain is apparently necessary for protein-protein interactions with constitutive and alternative splicing factors to stabilize hU2AF65 on the pyrimidine tract (38, 45). In contrast to the essential roles assigned to the two U2AF RS domains in vitro, molecular genetic analysis of the Drosophila U2AF RS domains indicates that either one of the RS domains is dispensable in vivo (19). Importantly, at least one RS domain on U2AF is essential for viability (19). The observation that the dU2AF38 RS domain is not essential in vivo (19) refocused our attention on domains present in the U2AF small subunit that are phylogenetically conserved. In an exhaustive database search for proteins containing RRMs, some of the signature sequences of this motif were identified in hU2AF35 (2). These sequences are also present in the Drosophila and S. pombe small-subunit homologs (21, 36). Although some of the most conserved residues in the RNA recognition motif are present in the U2AF small subunits, the RNP-1 octamer is highly degenerate and the RNP-2 hexamer is absent. Since these defining submotifs and other conserved residues are not present in the U2AF small-subunit RRM, it was termed a degenerate RRM or pseudo-RRM (2). Degenerate RRMs have been identified in a collection of RNA binding proteins, including several of the SR proteins, the pyrimidine tract binding protein, and the large subunit of U2AF (10). The degenerate RRMs in SRp30a (ASF/SF2) (4, 46), pyrimidine tract-binding protein (15), and hU2AF65 (42) were all found to be required for high-affinity RNA binding. Two putative Zn2+ binding domains, one on either side of the pseudo-RRM, were recently identified in hU2AF35 in a database search (37). These Cys3His Zn2+ binding motifs are conserved in all three small subunit homologs. Though sequence-specific RNA binding has not been described for proteins that contain this type of Zn2+ binding motif, several proteins that have this domain are involved in RNA metabolism (37). The evolutionary conservation of the pseudo-RRM and the two Zn2+ binding motifs in all three U2AF small subunit homologs suggested to us that these domains are important for function. The lack of requirement for the dU2AF38 RS domain in vivo prompted us to search for novel biochemical activities associated with the small subunit. The phylogenetically conserved, degenerate RRM (2) and two Zn2+ binding motifs (37) in the small subunit suggested that it might participate in RNA binding. While we detected weak RNA binding activity for the Drosophila small subunit on its own, we found that when complexed with the large subunit, dU2AF pyrimidine tract binding affinity increased 20-fold. This increase in RNA binding activity was not specific to Drosophila U2AF; the human U2AF heterodimer bound RNA with 15-fold-higher affinity than hU2AF65. Surprisingly, removal of the dU2AF38 RS domain abolished the increase in binding activity of the dU2AF heterodimer, indicating that the RS domain is necessary for high-affinity binding. Deletion of the dU2AF50 RS domain (dU2AF50ΔRS) dramatically reduced RNA binding activity of the large-subunit monomer. High-affinity binding was restored when the dU2AF38 RS domain was supplied in trans to dU2AF50ΔRS. These data suggest that high-affinity RNA binding activity requires at least one RS domain on U2AF, which is consistent with the requirement for at least one RS domain in vivo.

The pre-mRNA splicing factor U2AF (U2 snRNP auxiliary factor) has an essential role in 3' splice site selection. U2AF binds the intron pyrimidine tract between the branchpoint and the 3' splice site and recruits U2 snRNP to the branch site at an early step in spliceosome assembly. Human U2AF is a heterodimer composed of large (hU2AF65) and small (hU2AF35) subunits. Both subunits contain a domain enriched in arginine-serine dipeptide repeats termed an RS domain. The two U2AF RS domains have been assigned essential and independent roles in spliceosome assembly in vitro-the hU2AF65 RS domain is required to target U2 snRNP to the branch site and the hU2AF35 RS domain is necessary for protein-protein interactions with constitutive and alternative splicing factors. We have investigated the functional requirements for the RS domains on the Drosophila U2AF homolog in vivo. In sharp contrast to its essential role in U2 snRNP recruitment in vitro, the RS domain on the Drosophila large subunit homolog (dU2AF50) was completely dispensable in vivo. Prompted by this unexpected result, we analyzed the RS domain on the Drosophila small subunit homolog (dU2AF38). Despite its requirement for enhancer-dependent splicing activity in vitro, the dU2AF38 RS domain was also inessential in vivo. Finally, we have tested whether the Drosophila U2AF heterodimer requires any RS domain. Flies mutant for both the small and large subunits could not be rescued by dU2AF50deltaRS and dU2AF38deltaRS transgenes. Therefore, in contrast to the separate roles assigned to the U2AF RS domains in vitro, our genetic data suggest that they may have redundant functions in vivo.

The Drosophila melanogaster hnRNP protein, hrp48, is an abundant heterogeneous nuclear RNA-associated protein. Previous biochemical studies have implicated hrp48 as a component of a ribonucleoprotein complex involved in the regulation of the tissue-specific alternative splicing of the P-element third intron (IVS3). We have taken a genetic approach to analyzing the role of hrp48. Mutations in the hrp48 gene were identified and characterized. hrp48 is an essential gene. Hypomorphic mutations which reduce the level of hrp48 protein display developmental defects, including reduced numbers of ommatidia in the eye and morphological bristle abnormalities. Using a P-element third-intron reporter transgene, we found that reduced levels of hrp48 partially relieve IVS3 splicing inhibition in somatic cells. This is the first direct evidence that hrp48 plays a functional role in IVS3 splicing inhibition.

In mammalian cells, the Ku heterodimer is involved in DNA double-strand-break recognition and repair. We have established in yeast a connection between Ku activity and DNA double-strand-break damage repair, and a connection between Ku activity and commitment to DNA replication. We generated double-stranded DNA breaks in yeast cells in vivo by expressing a restriction endonuclease and have shown that yeast mutants lacking Ku p70 activity died while isogenic wild-type cells did not. Moreover, we have discovered that DNA damage occurs spontaneously during normal yeast mitotic growth, and that Ku functions in repair of this damage. We also observed that mitotically growing Ku p70 mutants have an anomalously high DNA content, suggesting a role for Ku in regulation of DNA synthesis. Finally, we present evidence that Ku p70 function is conserved between yeast, Drosophila , and humans.

When a large amount (milligram quantities) of RNA is desired (e.g., for making RNAs for RNA interference studies or for structural analyses), it is advantageous to use optimized transcription conditions. The method described here uses high millimolar concentrations of rNTPs, pyrophosphatase, and a specially formulated high-yield transcription buffer. These conditions prevent product (pyrophosphate) inhibition of RNA polymerase, which permits the accumulation of large quantities of RNA.

Transcription and pre-mRNA alternative splicing are highly regulated processes that play major roles in modulating eukaryotic gene expression. It is increasingly apparent that other pathways of RNA metabolism, including small RNA biogenesis, can regulate these processes. However, a direct link between alternative pre-mRNA splicing and small RNA pathways has remained elusive. Here we show that the small RNA pathway protein Argonaute-2 (Ago-2) regulates alternative pre-mRNA splicing patterns of specific transcripts in the Drosophila nucleus using genome-wide methods in conjunction with RNAi in cell culture and Ago-2 deletion or catalytic site mutations in Drosophila adults. Moreover, we show that nuclear Argonaute-2 binds to specific chromatin sites near gene promoters and negatively regulates the transcription of the Ago-2-associated target genes. These transcriptional target genes are also bound by Polycomb group (PcG) transcriptional repressor proteins and change during development, implying that Ago-2 may regulate Drosophila development. Importantly, both of these activities were independent of the catalytic activity of Ago-2, suggesting new roles for Ago-2 in the nucleus. Finally, we determined the nuclear RNA-binding profile of Ago-2, found it bound to several splicing target transcripts, and identified a G-rich RNA-binding site for Ago-2 that was enriched in these transcripts. These results suggest two new nuclear roles for Ago-2: one in pre-mRNA splicing and one in transcriptional repression.

Drosophila P elements are mobile DNA elements that encode an 87-kDa transposase enzyme and transpositional repressor proteins. One of these repressor proteins is the 207-amino-acid KP protein which is encoded by a naturally occurring P element with an internal deletion. To study the molecular mechanisms by which KP represses transposition, the protein was expressed, purified, and characterized. We show that the KP protein binds to multiple sites on the ends of P-element DNA, unlike the full-length transposase protein. These sites include the high-affinity transposase binding site, an 11-bp transpositional enhancer, and, at the highest concentrations tested, the terminal 31-hp inverted repeats. The DNA binding domain was localized to the N-terminal 98 amino acids and contains a CCHC sequence, a potential metal binding motif. We also demonstrate that the KP repressor protein can dimerize and contains two protein-protein interaction regions and that this dimerization is essential for high-affinity DNA binding.

During the past year, significant advances have been made in the field of pre-mRNA splicing. It is now clear that members of the serine-arginine-rich protein family are key players in exon definition and function at multiple steps in the spliceosome cycle. Novel findings have been made concerning the role of exon sequences, which function as both constitutive and regulated enhancers of splicing, in trans-splicing and as targets for tissue-specific control of splicing patterns. By combining biochemical approaches in human and yeast extracts with genetic analysis, much has been learned about the RNA-RNA and RNA-protein interactions that are necessary to assemble the various complexes that are found along the pathway to the catalytically active spliceosome.

Mobilizing Transposable Elements Transposable elements are parasitic DNA elements that can jump around the genome. They make up large portions of eukaryotic genomes (up to 50% of the human genome). In the short term, transposable elements can generate mutations and cause large-scale damage; but, in the long term, they can drive evolution. Majumdar et al. (p. 446 ) show that the human THAP9 gene (hTh9), which contains a THAP DNA binding domain also found in the Drosophila P -element transposase, is capable of mobilizing P -elements in Drosophila cells. The hTH9 gene also caused the excision of plasmid-borne Drosophila P -elements in human cells. Furthermore, hTh9 is able to nick P -element DNA and direct its transposition into the human genome.

Alternative splicing of pre-mRNA is a highly regulated process that allows cells to change their genetic informational output. These changes are mediated by protein factors that directly bind specific pre-mRNA sequences. Although much is known about how these splicing factors regulate pre-mRNA splicing events, comparatively little is known about the regulation of the splicing factors themselves. Here, we show that the Drosophila splicing factor P element Somatic Inhibitor (PSI) is phosphorylated at at least two different sites by at minimum two different kinases, casein kinase II (CK II) and tousled-like kinase (tlk). These phosphorylation events may be important for regulating protein-protein interactions involving PSI. Additionally, we show that PSI interacts with several proteins in Drosophila S2 tissue culture cells, the majority of which are splicing factors.

PSI is an RNA-binding protein involved in repressing splicing of the P element third intron in Drosophila somatic cell extracts. PSI produced in bacteria restores splicing inhibition to an extract relieved of inhibitory activity, indicating that PSI plays a direct role in somatic inhibition. Sequence analysis of cDNAs encoding PSI reveals three KH RNA-binding domains, a conserved motif also found in the yeast splicing regulator MER1. Notably, PSI is expressed highly in somatic embryonic nuclei but is undetectable in germ-line cells. In contrast, hrp48, another protein implicated in somatic inhibition, is found in the nucleus and cytoplasm of both tissues. The splicing inhibitory properties and soma-specific expression of PSI may be sufficient to explain the germ-line-specific transposition of P elements.

The large subunit of the human pre-messenger RNA splicing factor U2 small nuclear ribonucleoprotein auxiliary factor (hU2AF65) is required for spliceosome assembly in vitro. A complementary DNA clone encoding the large subunit of Drosophila U2AF (dU2AF50) has been isolated. The dU2AF50 protein is closely related to its mammalian counterpart and contains three carboxyl-terminal ribonucleoprotein consensus sequence RNA binding domains and an amino-terminal arginine- and serine-rich (R/S) domain. Recombinant dU2AF50 protein complements mammalian splicing extracts depleted of U2AF activity. Germline transformation of Drosophila with the dU2AF50 complementary DNA rescues a lethal mutation, establishing that the dU2AF50 gene is essential for viability. R/S domains have been found in numerous metazoan splicing factors, but their function is unknown. The mutation in Drosophila U2AF will allow in vivo analysis of a conserved R/S domain-containing general splicing factor.

Over the past year, significant progress has been made in the understanding of how RNA-binding factors may facilitate splice-site selection and spliceosome assembly, and confer fidelity to the pre-mRNA splicing reaction. In addition, a number of studies have revealed a complex network of RNA-RNA interactions in the spliceosome, strengthening the structural and functional parallels between nuclear pre-mRNA splicing and the self-splicing group I and group II introns. These new data further support the idea that pre-mRNA splicing occurs by RNA-mediated catalysis and illustrate quite dramatically the dynamic nature of conformational changes in the spliceosome cycle. With respect to tissue-specific pre-mRNA splicing, a number of studies have begun to illuminate mechanisms underlying control of splice-site selection and how so-called 'general' RNA-binding proteins, such as heterogeneous nuclear ribonucleoproteins, may be involved in determining different splicing patterns. Finally, an emerging theme involving the role of splicing in development is that differential transcriptional programs can be triggered in different cell types by alternative splicing patterns that generate transcription factor isoforms with different activities or DNA-binding specificities.

Splicing factor 1 (SF1) binds to the branch point sequence (BPS) of mammalian introns and is believed to be important for the splicing of some, but not all, introns. To help identify BPSs, particularly those that depend on SF1, we generated a BPS profile model in which SF1 binding affinity data, validated by branch point mapping, were iteratively incorporated into computational models. We searched a data set of 117,499 human introns for best matches to the SF1 Affinity Model above a threshold, and counted the number of matches at each intronic position. After subtracting a background value, we found that 87.9% of remaining high-scoring matches identified were located in a region upstream of 3'-splice sites where BPSs are typically found. Since U2AF65 recognizes the polypyrimidine tract (PPT) and forms a cooperative RNA complex with SF1, we combined the SF1 model with a PPT model computed from high affinity binding sequences for U2AF65. The combined model, together with binding site location constraints, accurately identified introns bound by SF1 that are candidates for SF1-dependent splicing.

The spliceosome, a dynamic assembly of proteins and RNAs, catalyzes the excision of intron sequences from nascent mRNAs. Recent work has suggested that the activity and composition of the spliceosome are regulated by ubiquitination, but the underlying mechanisms have not been elucidated. Here, we report that the spliceosomal Prp19 complex modifies Prp3, a component of the U4 snRNP, with nonproteolytic K63-linked ubiquitin chains. The K63-linked chains increase the affinity of Prp3 for the U5 snRNP component Prp8, thereby allowing for the stabilization of the U4/U6.U5 snRNP. Prp3 is deubiquitinated by Usp4 and its substrate targeting factor, the U4/U6 recycling protein Sart3, which likely facilitates ejection of U4 proteins from the spliceosome during maturation of its active site. Loss of Usp4 in cells interferes with the accumulation of correctly spliced mRNAs, including those for α-tubulin and Bub1, and impairs cell cycle progression. We propose that the reversible ubiquitination of spliceosomal proteins, such as Prp3, guides rearrangements in the composition of the spliceosome at distinct steps of the splicing reaction.

INTRODUCTIONDNA contamination of RNA preparations can present significant problems, especially for polymerase chain reaction (PCR)-based applications. No RNA extraction procedure excludes DNA entirely. Cytoplasmic RNA can be contaminated with DNA because of nuclei breakage during preparation. Moreover, TRIzol preparations do not exclude plasmids or other small DNA fragments. This article describes the reliable and effective method of eliminating DNA from RNA preparations via DNase digestion. DNase I specifically digests DNA into small oligonucleotides, leaving RNA intact.

Somatic inhibition restricts splicing of the Drosophila P-element third intron (IVS3) to the germ line. We have exploited this simple system to provide a model for a mechanism of alternative pre-mRNA splicing. Biochemical complementation experiments revealed that Drosophila somatic extracts inhibited U1 snRNP binding to the 5' splice site. Using sensitive RNase protection and modification-interference assays, we found that U1 snRNP bound to a pseudo-5' splice site in the 5' exon and that multiprotein complexes bound to an adjacent site. Binding of these factors appeared to mediate the inhibitory effect, because mutations in the pseudo-5' splice sites blocked binding and activated splicing in vitro. Likewise, wild-type, but not mutant, 5' exon RNA titrated inhibitory factors away from the pre-mRNA and activated splicing. Thus, we have defined the pseudo-5' splice sites as crucial components of the regulatory element, correlated the inhibitory activity with specific RNA binding factors from Drosophila somatic cells, and provided a mechanistic description of somatic inhibition. Because the inhibitory activity involves general splicing functions such as protein recognition of 5' splice site sequences and changes in the distribution of bound U1 snRNP, our data may also provide insights into how splice sites are selected.